Project Summary Ex vivo genetic modification of autologous CD34+ Hematopoietic stem/progenitor cells (HSPC) is a proven route to durable correction of blood diseases such as sickle cell disease, hemophilia, and HIV. At the core of these therapeutic approaches is delivery of exogenous genes or gene-modifying elements to HSPC. Current delivery methods (viral delivery or electroporation) are a major limitation to the widespread adoption of HSPC cell therapy due to their high cost, variable or limited effectiveness, and difficulty to scale up during development. Thus the pharmaceutical industry has a clear and urgent need for new delivery systems. These systems must be compact and enclosed, scalable from research to patient scale, and must preserve the viability and engraftment potential of the modified HSPC. Furthermore, such a system may be readily integrated into a point-of-care (POC) or even bedside manufacturing system. We have recently demonstrated a novel non-viral approach for efficient delivery of gene products through microfluidic mechanoporation that meets these design requirements. In this process, cells undergo a series of rapid and large intensity compressions to transiently open membrane pores and to induce active transport of target molecules into the cell interior. The advantage of this innovative approach for microfluidic intracellular delivery is large macromolecules (>2 MDa) are transported to the cells with high efficiency (70-90%), high cell viability (95+%), fast processing speed (100M cells/hr), and overall processing simplicity (few skilled steps). These outcomes have been met in proof-of-concept studies with cancer cell lines. The goal of the proposed work is to reproduce these studies in HSPC, and thus demonstrate feasibility of our device as a route to HSPC cell therapy manufacturing. The results of this study will define and validate microfluidic mechanotransfection as the first truly scalable (1 million cells to 1 billion cells using same process conditions) and POC cell therapy manufacturing technology.